CN101781667B - A kind of method utilizing wheat straw/corn stalk to produce bacterial cellulose - Google Patents

Abstract

The invention relates to a method for producing bacterial cellulose with wheat straws/maize straws, comprising the following steps: (1) grinding the wheat straws or maize straws, soaking the ground wheat straws or maize straws in dilute sulfuric acid or hydrochloric acid to react, then separating the residues of the wheat straws or maize straws from the acid hydrolysate through pump filtration and collecting the hydrolysate for later use; (2) detoxicating the hydrolysate; and (3) taking the detoxicated hydrolysate as the carbon source in the culture medium and adding the nitrogen source to prepare the culture medium and inoculating acetobacter aceti or gluconobacter oxydans into a shaker at 25-30 DEG C and 160-250r/min to be cultured or an incubator at 25-30 DEG C to undergo static culture, thus obtaining the bacterial cellulose. The carbon source in the culture medium is good in quality, low in price and suitable for industrial production.

Description

A kind of method utilizing wheat straw/corn stalk to produce bacterial cellulose

technical field

The invention belongs to the field of bacterial cellulose preparation, in particular to a method for producing bacterial cellulose by utilizing wheat straw/corn stalk.

Background technique

Bacterial Cellulose (BC) is also known as Microbial Cellulose. Compared with other higher plant cellulose in nature, it has many unique properties, including high purity, high crystallinity, and high degree of polymerization. , high water holding capacity, high tensile strength, strong biological adaptability, etc., can be widely used in artificial skin and blood vessels, medical dressings, adhesives, audio equipment vibration membranes, papermaking, textiles, composite membranes and other fields. Biomaterials with promising commercial applications. However, in the popularization and application of bacterial cellulose, problems such as high cost, low cellulose output and yield have become the main limiting factors for its industrial production and popularization and application.

Crop stalks such as wheat stalks and corn stalks are a huge amount of renewable biomass resources on the earth. my country is rich in biomass resources, and the total amount of biomass produced every year is more than 5 billion tons (dry weight). Therefore, the resource utilization of crop straw is a systematic project and a subject that needs to be developed urgently. The main components of wheat straw and corn straw are cellulose, hemicellulose and lignin. Among them, fiber and hemifiber are mainly composed of monosaccharides such as glucose and xylose, which are carbon sources that microorganisms can use. Finding an advanced and practical technology to convert the cellulose and hemicellulose into sugars that can be easily used by microorganisms, and to produce high value-added products through microbial fermentation, has great practical significance for solving environmental pollution and utilizing rice straw resources.

Contents of the invention

The technical problem to be solved by the present invention is to provide a method for producing bacterial cellulose by utilizing wheat stalks/corn stalks. The medium carbon source prepared by the method is of good quality and low price, and is suitable for industrial production.

A method for producing bacterial cellulose from wheat straw according to the present invention, comprising:

(1) Dilute acid hydrolysis of wheat straw

The wheat straw is first ground with a plant grinder, and then dilute sulfuric acid or hydrochloric acid (0.3% to 7%, w/v) is used in a reactor to form a solid/liquid ratio of 1:6-1:30 of wheat straw and dilute acid solution. Soak overnight (12-24h), then react at a temperature of 100°C-121°C for 30-80 minutes, then separate the straw residue and acid hydrolyzate by suction filtration, collect the hydrolyzate, and refrigerate at 4°C-8°C spare;

(2) Detoxification of hydrolyzate

Since the hydrolyzate contains certain toxic substances, Acetobacter aceti or Gluconobacter Oxydans cannot grow and synthesize bacterial cellulose when the hydrolyzate is used as the carbon source of the medium, so the hydrolyzate must be detoxified;

Use alkalis such as NaOH, Ca(OH) ₂ (or lime) and ammonia water (NH ₄ OH) to detoxify the hydrolyzate, change the detoxification conditions such as pH value, time, and combine with activated carbon or 10% laccase (enzyme activity is 2.75U/mL) to detoxify the hydrolyzate to improve the detoxification effect;

Method 1: NaOH adjusts the pH value of the hydrolyzate to 4.5-5.5, filters the precipitate, and fine-tunes it to pH 4.5-5.5;

Method 2: Adjust the pH value of the hydrolyzate to 4.5-5.5 with NaOH, add activated carbon for adsorption, filter out the activated carbon after the reaction and fine-tune the pH value to 4.5-5.5;

Method 3: Adjust the pH value of the hydrolyzate to 9.5-11 with NaOH, add activated carbon for adsorption, filter out the activated carbon after the reaction and re-adjust the pH value to 4.5-5.5;

Method 4: Adjust the pH value of the hydrolyzate to 9.5-11 with NaOH, react overnight in a warm water bath at 25-60°C, filter and re-adjust the pH value to 4.5-5.5;

Method 5: Adjust the pH value of the hydrolyzate to 9.5-11 with NaOH, react overnight in a warm water bath at 25-60°C, filter and re-adjust the pH value to 4.5-5.5, then add activated carbon for adsorption, filter out the activated carbon after the reaction and re- Fine-tune the pH value to 4.5-5.5;

Method 6: Adjust the pH value of the hydrolyzate to 4.5-5.5 with NaOH, add 10% laccase (enzyme activity: 2.75U/mL) and react in a warm water bath at 25-60°C for 12h-24h, filter out the precipitate and fine-tune again pH to 4.5-5.5;

Method 7: Ca(OH) ₂ adjusts the pH value of the hydrolyzate to 4.5-5.5, filters the precipitate, and fine-tunes it to pH 4.5-5.5;

Method 8: Adjust the pH value of the hydrolyzate to 4.5-5.5 with Ca(OH) ₂ , add activated carbon for adsorption, filter out the activated carbon after the reaction and fine-tune the pH value to 4.5-5.5;

Method 9: Ca(OH) ₂ adjust the pH value of the hydrolyzate to 9.5-11, add activated carbon for adsorption, filter out the activated carbon after the reaction and re-adjust the pH value to 4.5-5.5;

Method 10: Ca(OH) ₂ adjusts the pH value of the hydrolyzate to 9.5-11, reacts overnight in a warm water bath at 25-60°C, filters and readjusts the pH value to 4.5-5.5;

Method 11: Ca(OH) ₂ Adjust the pH value of the hydrolyzate to 9.5-11, react overnight in a warm water bath at 25-60°C, filter and re-adjust the pH value to 4.5-5.5, then add activated carbon for adsorption, filter after reaction Remove the activated carbon and re-fine-tune the pH to 4.5-5.5;

Method 12: Ca(OH) ₂ Adjust the pH value of the hydrolyzate to 4.5-5.5, add 10% laccase (enzyme activity: 2.75U/mL) and react in a warm water bath at 25-60°C for 12h-24h, filter out the precipitate and re-adjust the pH value to 4.5-5.5;

Method 13: Adjust the pH value of the hydrolyzate to 9.5-11 with 25%-30% ammonia water, react overnight in a warm water bath at 25-60°C, filter and re-adjust the pH value to 4.5-5.5, then add activated carbon for adsorption, after the reaction Filter out the activated carbon and re-fine-tune the pH to 4.5-5.5;

Method 14: 25%-30% ammonia water to adjust the pH value of the hydrolyzate to 4.5-5.5, add 10% laccase (enzyme activity is 2.75U/mL) and react in a warm water bath at 25-60°C for 12h-24h, filter out Precipitate and re-fine-tune pH to 4.5-5.5.

(3) Preparation of bacterial cellulose

Take the above-mentioned detoxified hydrolyzate as the carbon source of the medium, add 0.1-1% yeast extract and 0.1-0.5% tryptone to make a medium, and insert Acetobacter acetobacter (美国标准生物样品保藏中心ATCC提供：Acetobacter aceti subsp.xylinus(Gluconacetobacter xylinus)ATCC 23770、Acetobacteraceti subsp.xylinus(Gluconacetobacter xylinus)ATCC 53263、Gluconacetobacter xylinusATCC 53264、Gluconacetobacter xylinus ATCC 53524、Gluconacetobacter xylinus ATCC53582、Gluconacetobacter xylinus ATCC 53749、 Gluconacetobacter xylinus ATCC 53750, Gluconacetobacter xylinus ATCC 700178, Gluconacetobacter hansenii ATCC 10821, Gluconacetobacter hansenii ATCC 23769) or Gluconobacter oxydans (Gluconobacter oxydans ATCC11894) and other bacteria in 25-30 ℃ shaker or 25-30 ℃ for 0/min, 16 Static culture in -30°C incubator, after a period of time (6-25 days), ideal bacterial cellulose can be obtained.

The wheat straw in the step (1) is first ground to 40 mesh with a plant pulverizer.

The activated carbon in the step (2) is 1%-6% by mass of activated carbon and stirred at room temperature for 5-10min.

The nitrogen source in the step (3) is one or more of ammonium salts such as yeast extract, peptone, tryptone, beef extract, and ammonium sulfate.

The carbon source used in the step (3) for cultivating bacteria is the detoxified wheat straw hydrolyzate, and the hydrolyzate is prepared into a fermentation medium according to a reducing sugar amount of 7-30g/L (calculated as glucose), containing 0.1 -1% yeast extract, 0.1-0.5% peptone, medium pH 5.0.

The inoculation amount in the step (3) is 6%.

Among them, method 11, the method of combining Ca(OH) ₂ with activated carbon to prepare bacterial cellulose has the best effect, and can generate a relatively ideal bacterial cellulose film in about 11 days, and the bacterial cellulose yield is the highest, up to 15.4mg/ mL. When the detoxified hydrolyzate was compared with other conventional carbon sources, under the same carbon source concentration and culture conditions, the yield of bacterial cellulose prepared by the detoxified hydrolyzate was still higher than that prepared by conventional carbon sources, compared with pure mannitol , sucrose or glucose as carbon source, the cellulose yield was 50.3%, 65.0% and 69.9% higher, respectively.

In addition, the method of using different lyes to adjust the pH of the hydrolyzate to 9.5-11 and then combining activated carbon (method 5, method 11, method 13) is better than adjusting the pH of the hydrolyzate to 4.5-5.5 with different lyes and then combining laccase Methods (method 6, method 12, and method 14) have good detoxification effects on wheat straw hydrolyzate, and the best detoxification effect is to adjust the pH value of hydrolyzate with Ca(OH) ₂ , followed by NaOH and ammonia water.

The main chemical components of wheat straw are composed of cellulose, hemicellulose and lignin, but the structure of wheat straw is complex. Cellulose and hemicellulose are not only wrapped by lignin, but also partially covalently bonded with lignin, and cellulose has a highly ordered crystal structure, so the utilization of wheat straw requires pretreatment. Only after pretreatment can the encapsulation of lignin on cellulose be released, thereby exposing the cellulose, which is beneficial to acid hydrolysis or enzyme hydrolysis and subsequent fermentation process. During the hydrolysis process, although mixed sugars such as glucose, xylose, and arabinose are produced, due to the severe reaction conditions, many inhibitors that are toxic to fermenting microorganisms will be generated. The inhibitors in the hydrolyzate mainly include: furfural, Hydroxymethylfurfural, acetic acid, phenolic compounds, syringic acid, hydroxybenzoic acid, vanillin and other toxic compounds. Therefore, in the process of microbial fermentation using the above-mentioned hydrolyzate, it is necessary to detoxify the hydrolyzate to reduce the influence of these toxic compounds on the microbial fermentation culture.

A kind of method utilizing corn stalk to produce bacterial cellulose of the present invention comprises:

(1) Dilute acid hydrolysis of corn stalks

Air-dried and pulverized corn stalks, soaked them with 1% to 8% (w/v) sulfuric acid or hydrochloric acid, the solid-liquid ratio of corn stalks to sulfuric acid or hydrochloric acid was 1:5 to 1:25, and then heated at 90°C to 140°C React for 10-90 minutes, after the reaction is completed, filter with suction to collect the hydrolyzate;

(2) Detoxification of hydrolyzate

Use NaOH, Ca(OH) ₂ (or lime), ammonia water (NH ₄ OH) and other alkalis to adjust the pH value of the hydrolyzate, and combine activated carbon or laccase to treat the hydrolyzate.

Method 1: Use NaOH to adjust the pH value of the hydrolyzate to 4-6, centrifuge or filter to remove the precipitate;

Method 2: Use NaOH to adjust the pH value of the hydrolyzate to 4-6, add activated carbon to react for 5min-30min, centrifuge or filter to remove the precipitate;

Method 3: Use NaOH to adjust the pH value of the hydrolyzate to 9-11, add activated carbon to react for 5min-30min, centrifuge or filter to remove the precipitate, and adjust the pH value of the hydrolyzate to 4-6;

Method 4: Use NaOH to adjust the pH value of the hydrolyzate to 9-11, react in a water bath at 20-60°C for 12-24 hours, centrifuge or filter to remove the precipitate, and adjust the pH value of the hydrolyzate to 4-6;

Method 5: Use NaOH to adjust the pH value of the hydrolyzate to 9-11, react in a water bath at 20-60°C for 12-24 hours, adjust the pH value of the hydrolyzate to 4-6, then add activated carbon to react for 5min-30min, centrifuge or filter to remove the precipitate;

Method 6: Use NaOH to adjust the pH value of the hydrolyzate to 4-6, centrifuge or filter to remove the precipitate, add 10% laccase with an enzyme activity of 2.75 U/mL, and react in a water bath at 30-60°C for 12-24 hours;

Method 7: Use Ca(OH) ₂ to adjust the pH value of the hydrolyzed solution to 4-6, centrifuge or filter to remove the precipitate;

Method 8: Use Ca(OH) ₂ to adjust the pH value of the hydrolyzate to 4-6, add activated carbon to react for 5min-30min, centrifuge or filter to remove the precipitate;

Method 9: Use Ca(OH) ₂ to adjust the pH value of the hydrolyzate to 9-11, add activated carbon to react for 5min-30min, centrifuge or filter to remove the precipitate, and adjust the pH value of the hydrolyzate to 4-6;

Method 10: Use Ca(OH) ₂ to adjust the pH value of the hydrolyzate to 9-11, react in a water bath at 20-60°C for 12-24 hours, centrifuge or filter to remove the precipitate, and adjust the pH value of the hydrolyzate to 4-6;

Method 11: Use Ca(OH) ₂ to adjust the pH value of the hydrolyzate to 9-11, react in a water bath at 20-60°C for 12-24 hours, adjust the pH value of the hydrolyzate to 4-6, and then add activated carbon to react for 5min- 30min, centrifuge or filter to remove precipitate;

Method 12: Use Ca(OH) ₂ to adjust the pH value of the hydrolyzate to 4-6, centrifuge or filter to remove the precipitate, add laccase and react in a water bath at 30-60°C for 12-24 hours;

Method 13: Use ammonia water to adjust the pH value of the hydrolyzate to 9-11, react in a water bath at 20-60°C for 12-24 hours, adjust the pH value of the hydrolyzate to 4-6, then add activated carbon to react for 5min-30min, centrifuge or filter to remove the precipitate;

Method 14: Adjust the pH value of the hydrolyzate to 4-6 with ammonia water, centrifuge or filter to remove the precipitate, add 10% laccase with an enzyme activity of 2.75 U/mL, and react in a water bath at 30-60°C for 12-24 hours;

(3) Take the above-mentioned detoxified hydrolyzate as the carbon source of the culture medium, add 0.1-2% nitrogen source, and use it as a culture medium after sterilizing at 121° C. for 15 minutes; insert Acetobacter acetobacter (美国标准生物样品保藏中心ATCC提供：Acetobacter aceti subsp.xylinus(Gluconacetobacter xylinus)ATCC 23770、Acetobacter acetisubsp.xylinus(Gluconacetobacter xylinus)ATCC 53263、Gluconacetobacter xylinus ATCC53264、Gluconacetobacter xylinus ATCC 53524、Gluconacetobacter xylinus ATCC 53582、Gluconacetobacter xylinus ATCC 53749 , Gluconacetobacter xylinus ATCC 53750, Gluconacetobacter xylinus ATCC 700178, Gluconacetobacter hansenii ATCC 10821, Gluconacetobacter hansenii ATCC 23769) or Gluconacetobacter oxydans ATCC 23769) or glucose oxidizing bacteria (Gluconobacter oxydans ATCC11894) and other bacteria, at 25 ~ 30 ℃, 0 5 pm, 0 Static culture for 6-25 days to obtain bacterial cellulose.

The activated carbon in the step (2) is 1%-6% by mass of activated carbon and stirred at room temperature for 5-10min.

The nitrogen source in the step (3) is one or more of ammonium salts such as yeast extract, peptone, tryptone, beef extract, and ammonium sulfate.

The medium of the step (3) adopts the detoxified hydrolyzate as the carbon source of the medium, and the hydrolyzate is formulated into a fermentation medium according to the reducing sugar amount (calculated as glucose) of 7-30g/L, containing 0.1-1% yeast Extract, 0.1-0.5% peptone, medium pH value 5.0.

The experimental results show that the hydrolyzed solution obtained by hydrolyzing corn stalks with 5% sulfuric acid at a solid-liquid ratio of 1:10 for 30 minutes was treated with Ca(OH) ₂ combined with activated carbon, and the sugar concentration of the hydrolyzed solution was adjusted to 20g/L, adding 0.5% Yeast extract and 0.5% peptone were formulated into culture medium, inoculated with Acetobacter acetobacter with 6% inoculum size, and cultured statically at 30°C for 10 days. The yield of bacterial cellulose was 15.6g/L, and the yield of bacterial cellulose was respectively It is 40.2%, 55.6% and 64.3% higher than when mannitol, sucrose and glucose are used as carbon sources.

Beneficial effect

(1) the present invention is simple, with low cost, and raw material source is extensive, is suitable for industrialized production;

(2) The present invention utilizes this cheap raw material in wheat straw, carries out dilute acid hydrolysis and detoxification, produces a kind of medium carbon source that can be used for cultivating bacteria to prepare cellulose, and is industrialized large-scale production bacterial cellulose This emerging biological material provides new ideas and approaches; wheat straw has a wide range of sources and is cheap, so the preparation of culture medium carbon source for bacterial cellulose production and its detoxification method have high practical application value and obvious advantages; after testing , the medium carbon source produced by the present invention can also be used for the cultivation of other industrial microorganisms, and is a low-cost and high-quality carbon source;

(3) Corn stalk is a kind of renewable resource with wide sources and low price. Utilizing the method of the present invention to process corn stalk can not only obtain a carbon source suitable for preparing bacterial cellulose, and provide a new way for the industrialized production of bacterial cellulose, but also The straw can be effectively used and the waste of resources can be reduced; the corn straw is used to prepare bacterial cellulose after acid hydrolysis and treatment, and the yield of bacterial cellulose is greatly increased, which is 55.6% and 64.3% higher than when sucrose and glucose are used as carbon sources, respectively.

Description of drawings

Figure 1 The results of bacterial cellulose produced from wheat straw hydrolyzate carbon source prepared by different methods.

Detailed ways

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Example 1

Wheat straw is first ground with a plant pulverizer, then soaked overnight (12-24h) with dilute sulfuric acid (3%, w/v) in a reactor at a solid/liquid ratio of 1:6 between the straw and dilute acid solution, Then react at a temperature of 121° C. for 60 minutes, then separate the straw residue and acid solution by suction filtration, collect the hydrolyzate, and refrigerate at 4° C. for later use.

Add NaOH to adjust the pH value of the hydrolyzed liquid to about 10, filter the precipitate with filter paper to obtain the treated hydrolyzed liquid, and then fine-tune the pH value to 10.0. Then seal it with a film, place it in a 30°C water bath for 12-24 overnight, and finally adjust the pH value of the hydrolyzate to 5.0 with dilute acid. Then add 2% (mass percentage) activated carbon, stir (at room temperature for 5-10min), filter out the activated carbon with filter paper to obtain detoxified hydrolyzed clear liquid, and fine-tune the pH value to 5.5 with dilute sulfuric acid. The detoxified hydrolyzate is tested for sugar and used as the carbon source of the medium, and then 0.1%-1% of yeast extract and 0.1%-0.5% tryptone are added to make the straw hydrolyzate medium for microbial growth nourish.

Example 2

Wheat straw is first ground with a plant grinder, and then soaked overnight (12-24h) in a reactor with dilute hydrochloric acid (1%, w/v) at a solid/liquid ratio of 1:10 straw to dilute acid solution, Then react at a temperature of 121° C. for 75 minutes, then separate the straw residue and acid solution by suction filtration, collect the hydrolyzate, and refrigerate at 4° C. for later use.

Add Ca(OH) ₂ to adjust the pH value of the hydrolyzed liquid to about 10, filter the precipitate with filter paper to obtain the treated hydrolyzed liquid, and then fine-tune the pH value to 10.0. Then seal it with a film, place it in a 40°C water bath for 12-24 overnight, and finally adjust the pH value of the hydrolyzate to 5.0 with dilute acid. Then add 2% (mass percentage) activated carbon, stir (at room temperature for 5-10min), filter out the activated carbon with filter paper to obtain detoxified hydrolyzed clear liquid, and fine-tune the pH value to 5.5 with dilute sulfuric acid. The detoxified hydrolyzate is tested for sugar and used as the carbon source of the medium, and then 0.1%-1% of yeast extract and 0.1%-0.5% tryptone are added to make the straw hydrolyzate medium for microbial growth nourish.

Example 3

Wheat straw is first ground with a plant pulverizer, and then soaked overnight (12-24h) with dilute sulfuric acid (2%, w/v) in a reactor with a solid/liquid ratio of 1:15 straw to dilute acid solution, Then react at a temperature of 100° C. for 80 minutes, then separate the straw residue and acid solution by suction filtration, collect the hydrolyzate, and refrigerate at 4° C. for later use.

Add 25% ammonia water to adjust the pH value of the hydrolyzed liquid to 10, filter out the precipitate with filter paper to obtain the treated hydrolyzed liquid, and fine-tune the pH value to 10. Then seal it with a film, place it in a 25°C water bath to react overnight, and finally adjust the pH value of the hydrolyzate to 5.0 with dilute acid. Then add 2% (mass percentage) activated carbon, stir (at room temperature for 5-10min), filter out the activated carbon with filter paper to obtain detoxified hydrolyzed clear liquid, and fine-tune the pH value to 5.5 with dilute sulfuric acid. The detoxified hydrolyzate is tested for sugar and used as the carbon source of the medium, and then 0.1%-1% of yeast extract and 0.1%-0.5% tryptone are added to make the straw hydrolyzate medium for microbial growth nourish.

Example 4

Wheat straw is first ground with a plant pulverizer, then soaked overnight (12-24h) with dilute sulfuric acid (3%, w/v) in a reactor at a solid/liquid ratio of 1:15 between the straw and dilute acid solution, Then react at a temperature of 110° C. for 60 minutes, then separate the straw residue and acid solution by suction filtration, collect the hydrolyzate, and refrigerate at 6° C. for later use.

Add NaOH to adjust the pH value of the hydrolyzed supernatant to 5.0, add 10% laccase with an enzyme activity of 2.75 U/mL, react in a water bath at 50°C for 12 hours, filter out the activated carbon and fine-tune the pH value to 5.5 again. The detoxified hydrolyzate is tested for sugar and used as the carbon source of the medium, and then 0.1%-1% of yeast extract and 0.1%-0.5% tryptone are added to make the straw hydrolyzate medium for microbial growth nourish.

Example 5

Wheat straw is first ground with a plant pulverizer, and then soaked overnight (12-24h) with dilute sulfuric acid (3%, w/v) in a reactor at a solid/liquid ratio of 1:30 straw to dilute acid solution, Then react at a temperature of 110° C. for 60 minutes, then separate the straw residue and acid solution by suction filtration, collect the hydrolyzate, and refrigerate at 6° C. for later use.

Add Ca(OH) ₂ to adjust the pH value of the hydrolyzed supernatant to 5.0, add 10% laccase with an enzyme activity of 2.75 U/mL, react in a water bath at 50°C for 12 hours, filter out the activated carbon and fine-tune the pH value to 5.5 again. The detoxified hydrolyzate is tested for sugar and used as the carbon source of the medium, and then 0.1%-1% of yeast extract and 0.1%-0.5% tryptone are added to make the straw hydrolyzate medium for microbial growth nourish.

Example 6

The wheat straw is first ground with a plant pulverizer, and then soaked overnight (12-24h) with dilute hydrochloric acid (3%, w/v) in a reactor at a solid/liquid ratio of 1:30 wheat straw to dilute acid solution, Then react at a temperature of 121° C. for 60 minutes, then separate the straw residue and acid solution by suction filtration, collect the hydrolyzate, and refrigerate at 4° C. for later use.

Add 25% ammonia water to adjust the pH value of the hydrolyzed supernatant to 5.0, add 10% laccase with an enzyme activity of 2.75 U/mL, react in a water bath at 40°C for 12 hours, filter out activated carbon and fine-tune the pH value to 5.5 again. The detoxified hydrolyzate is tested for sugar and used as the carbon source of the medium, and then 0.1%-1% of yeast extract and 0.1%-0.5% tryptone are added to make the straw hydrolyzate medium for microbial growth nourish.

Example 7

Wheat straw is first ground with a plant pulverizer, and then soaked overnight (12-24h) with dilute sulfuric acid (1%, w/v) in a reactor at a solid/liquid ratio of 1:12 between the straw and dilute acid solution, Then react at a temperature of 121° C. for 30 minutes, then separate the straw residue and acid solution by suction filtration, collect the hydrolyzate, and refrigerate at 4° C. for later use.

Add Ca(OH) ₂ to adjust the pH value of the hydrolyzed liquid to about 10, filter the precipitate with filter paper to obtain the treated hydrolyzed liquid, and then fine-tune the pH value to 10.0. Then seal it with a film, place it in a 40°C water bath to react overnight, and finally adjust the pH value of the hydrolyzate to 5.0 with dilute acid. Then add 2% (mass percentage) activated carbon, stir (at room temperature for 5-10min), filter out the activated carbon with filter paper to obtain detoxified hydrolyzed clear liquid, and fine-tune the pH value to 5.5 with dilute sulfuric acid. The detoxified hydrolyzate is tested for sugar and used as the carbon source of the medium, and then 0.1%-1% of yeast extract and 0.1%-0.5% tryptone are added to make the straw hydrolyzate medium for microbial growth nourish.

Example 8

Use the above-mentioned various methods to detoxify the wheat straw hydrolyzate, and adjust the sugar concentration of the hydrolyzate to 25g/L. The paste and 0.1%-0.5% tryptone were formulated into 50mL wheat straw hydrolyzate medium, glucose medium, mannitol medium and sucrose medium respectively. Inoculate 6-10% inoculum of Acetobacter or Glucobacterium Oxidus into wheat straw hydrolyzate medium and culture statically in a 30°C incubator for 8-15 days to obtain ideal bacterial cellulose products or more abundant bacteria Cellulose membrane, the experimental results are shown in Figure 1.

The detoxification method using Ca(OH) _2- bound activated carbon (Method 11) was superior to those using NaOH-bound activated carbon and ammonia-bound activated carbon in terms of BC yield. The carbon source prepared by the detoxification method of Ca(OH) ₂ combined with activated carbon can form an ideal bacterial cellulose film in about 8-15 days, while the carbon source prepared by the detoxification method of NaOH combined with activated carbon and ammonia water combined with activated carbon Source, although it can also form bacterial cellulose film, but the yield is not as high as the detoxification method of Ca(OH) ₂ combined with activated carbon.

It can be seen from Fig. 1 that the detoxification method of Ca(OH) ₂ combined with activated carbon (method 11) yielded the highest BC yield when Ca(OH) ₂ combined with activated carbon and other conventional carbon sources such as sucrose, glucose and mannitol When compared, Ca(OH) ₂ combined with activated carbon detoxified hydrolyzate produced higher bacterial cellulose yield than medium with conventional carbon source. Therefore, under the same conditions, the yield of bacterial cellulose produced by the medium prepared with detoxified wheat straw hydrolyzate is slightly higher than that of the medium with mannitol, sucrose or glucose as carbon source. Due to the wide source of raw material wheat straw and low price, Therefore, the preparation of the medium carbon source for bacterial cellulose production and its detoxification method have high practical application value and obvious advantages.

Example 9

After the corn stalks were air-dried and pulverized, 3% (w/v) sulfuric acid was added to the reactor at a solid-to-liquid ratio of 1:10, and then reacted at 100°C for 80 minutes. After the reaction, the residue and the hydrolyzed solution were separated by suction filtration and collected. Hydrolyzate.

Use Ca(OH) ₂ to adjust the pH value of the hydrolyzed solution to 10, react in a water bath at 30°C for 12-24 hours, adjust the pH value of the hydrolyzed solution to 5, then add activated carbon to react for 30 minutes, centrifuge or filter to remove the precipitate;

The above treated hydrolyzate is used as carbon source, supplemented with 0.1% yeast extract and 0.5% peptone, and used as culture medium after sterilization. Inoculate the bacterial species with an inoculum amount of 6%, vibrate culture or static culture at 30° C. and 160-250 rpm for 10 days to obtain bacterial cellulose.

Example 10

After the corn stalks were air-dried and pulverized, 5% (w/v) hydrochloric acid was added to the reactor at a solid-to-liquid ratio of 1:15, and then reacted at 100°C for 60 minutes. After the reaction, the residue and the hydrolyzed solution were separated by suction filtration, and collected Hydrolyzate.

Use Ca(OH) ₂ to adjust the pH value of the hydrolyzate to 5, centrifuge or filter to remove the precipitate, add laccase and react in a water bath at 35°C for 24 hours;

The above treated hydrolyzate is used as carbon source, supplemented with 0.1% yeast extract and 0.5% tryptone, and used as culture medium after sterilization. Inoculate the strain with 10% inoculum amount, shake culture or static culture at 30° C. and 160-250 rpm for 8 days to obtain bacterial cellulose.

Example 11

After the corn stalks were air-dried and pulverized, 1% (w/v) sulfuric acid was added to the reactor at a solid-to-liquid ratio of 1:8, and then reacted at 120°C for 90 minutes. After the reaction, the residue and the hydrolyzed solution were separated by suction filtration and collected. Hydrolyzate.

Use NaOH to adjust the pH value of the hydrolyzate to 5.5, add activated carbon to react for 30 minutes, centrifuge or filter to remove the precipitate;

The above treated hydrolyzate is used as carbon source, supplemented with 0.1% yeast extract and 0.5% peptone, and used as culture medium after sterilization. Inoculate the bacterial species with an inoculum amount of 8%, vibrate culture or static culture at 30° C. and 160-250 rpm for 12 days to obtain bacterial cellulose.

Example 12

After the corn stalks were air-dried and pulverized, 6% (w/v) sulfuric acid was added to the reactor at a solid-to-liquid ratio of 1:20, and then reacted at 90°C for 30 minutes. After the reaction, the residue and the hydrolyzed solution were separated by suction filtration and collected. Hydrolyzate.

Use NaOH to adjust the pH value of the hydrolyzate to 5, centrifuge or filter to remove the precipitate, add laccase and react in a water bath at 35°C for 24 hours;

The above treated hydrolyzate was used as carbon source, supplemented with 1% peptone, and sterilized as culture medium. Inoculate the bacterial species with an inoculum amount of 8%, vibrate culture or static culture at 30° C. and 160-250 rpm for 10 days to obtain bacterial cellulose.

Example 13

After the corn stalks were air-dried and pulverized, 5% (w/v) sulfuric acid was added to the reactor at a solid-to-liquid ratio of 1:25, and then reacted at 90°C for 45 minutes. After the reaction, the residue and the hydrolyzed solution were separated by suction filtration and collected. Hydrolyzate.

Use ammonia water to adjust the pH value of the hydrolyzate to 11, react in a water bath at 50°C for 12-24 hours, adjust the pH value of the hydrolyzate to 5, then add activated carbon to react for 30 minutes, centrifuge or filter to remove the precipitate;

The above treated hydrolyzate is used as carbon source, supplemented with 0.1% beef extract and 0.5% peptone, and used as a culture medium after sterilization. Bacterial cellulose was inoculated with 10% inoculum amount, shaken or static cultured at 30° C. and 160 rpm for 15 days to obtain bacterial cellulose.

Example 14

After the corn stalks were air-dried and pulverized, 3% (w/v) hydrochloric acid was added to the reactor at a solid-to-liquid ratio of 1:6, and then reacted at 120°C for 45 minutes. After the reaction, the residue and the hydrolyzed solution were separated by suction filtration and collected. Hydrolyzate.

Use NaOH to adjust the pH value of the hydrolyzed solution to 10, react in a water bath at 30°C for 12-24 hours, centrifuge or filter to remove the precipitate, and adjust the pH value of the hydrolyzed solution to 5;

The above treated hydrolyzate is used as carbon source, supplemented with 0.1% yeast extract and 0.5% peptone, and used as culture medium after sterilization. Bacterial cellulose was inoculated with 10% inoculation amount, shaken or static cultured at 30° C. and 160 rpm for 10 days to obtain bacterial cellulose.

Example 15

After the corn stalks were air-dried and pulverized, 1% (w/v) hydrochloric acid was added to the reactor at a solid-to-liquid ratio of 1:10, and then reacted at 140°C for 30 minutes. After the reaction, the residue and the hydrolyzed solution were separated by suction filtration and collected. Hydrolyzate.

Use Ca(OH) ₂ to adjust the pH value of the hydrolyzate to 11, react in a water bath at 30°C for 12-24 hours, centrifuge or filter to remove the precipitate, and adjust the pH value of the hydrolyzate to 5;

The above treated hydrolyzate is used as carbon source, supplemented with 0.1% beef extract and 0.5% peptone, and used as a culture medium after sterilization. Inoculate the bacterial species with an inoculum amount of 8%, vibrate culture or static culture at 30° C. and 160-250 rpm for 10 days to obtain bacterial cellulose.

Example 16

After the corn stalks were air-dried and pulverized, 5% (w/v) hydrochloric acid was added to the reactor at a solid-to-liquid ratio of 1:8, and then reacted at 100°C for 30 minutes. After the reaction, the residue and the hydrolyzed solution were separated by suction filtration and collected. Hydrolyzate.

Use Ca(OH) ₂ to adjust the pH value of the hydrolyzate to 10, add activated carbon to react for 30 minutes, centrifuge or filter to remove the precipitate, and adjust the pH value of the hydrolyzate to 5;

The above treated hydrolyzate is used as carbon source, supplemented with 0.1% yeast extract and 0.5% peptone, and used as culture medium after sterilization. Inoculate the bacterial species with an inoculum amount of 6%, and vibrate or static culture at 30° C. and 180 rpm for 15 days to obtain bacterial cellulose.

Example 17

After the corn stalks were air-dried and pulverized, 3% (w/v) sulfuric acid was added to the reactor at a solid-to-liquid ratio of 1:12, and then reacted at 120°C for 30 minutes. After the reaction, the residue and the hydrolyzed solution were separated by suction filtration and collected. Hydrolyzate.

Use Ca(OH) ₂ to adjust the pH value of the hydrolyzed solution to 5.5, centrifuge or filter to remove the precipitate;

The above treated hydrolyzate is used as carbon source, supplemented with 0.5% yeast extract and 0.5% peptone, and used as culture medium after sterilization. Bacterial cellulose was inoculated with 10% inoculum, shaken or static cultured at 30° C. and 200 rpm for 11 days to obtain bacterial cellulose.

Claims (3)

1. A method of utilizing corn stalks to produce bacterial cellulose, comprising: (1) Air-dry and pulverize the corn stalks, soak them in 5% to 8% w/v sulfuric acid or hydrochloric acid, the solid-to-liquid ratio of corn stalks to sulfuric acid or hydrochloric acid is 1:15 to 1:25, and then soak them at 90°C to 100°C The reaction was carried out for 10-90 minutes, and after the reaction was completed, it was suction filtered to collect the hydrolyzate; (2) Detoxification of hydrolyzate Method 1: Use NaOH to adjust the pH value of the hydrolyzate to 4-6, centrifuge or filter to remove the precipitate; Method 2: Use NaOH to adjust the pH value of the hydrolyzate to 4-6, add activated carbon to react for 5min-30min, centrifuge or filter to remove the precipitate; Method 3: Use NaOH to adjust the pH value of the hydrolyzate to 9-11, add activated carbon to react for 5min-30min, centrifuge or filter to remove the precipitate, and adjust the pH value of the hydrolyzate to 4-6; Method 4: Use NaOH to adjust the pH value of the hydrolyzate to 9-11, react in a water bath at 20-60°C for 12-24 hours, centrifuge or filter to remove the precipitate, and adjust the pH value of the hydrolyzate to 4-6; Method 5: Use NaOH to adjust the pH value of the hydrolyzate to 9-11, react in a water bath at 20-60°C for 12-24 hours, adjust the pH value of the hydrolyzate to 4-6, then add activated carbon to react for 5min-30min, centrifuge or filter to remove the precipitate; Method 6: Use NaOH to adjust the pH value of the hydrolyzate to 4-6, centrifuge or filter to remove the precipitate, add 10% laccase with an enzyme activity of 2.75U/mL, and react in a water bath at 30-60°C for 12-24 hours; Method 7: Use Ca(OH) ₂ to adjust the pH value of the hydrolyzed solution to 4-6, centrifuge or filter to remove the precipitate; Method 8: Use Ca(OH) ₂ to adjust the pH value of the hydrolyzate to 4-6, add activated carbon to react for 5min-30min, centrifuge or filter to remove the precipitate; Method 9: Use Ca(OH) ₂ to adjust the pH value of the hydrolyzate to 9-11, add activated carbon to react for 5min-30min, centrifuge or filter to remove the precipitate, and adjust the pH value of the hydrolyzate to 4-6; Method 10: Use Ca(OH) ₂ to adjust the pH value of the hydrolyzate to 9-11, react in a water bath at 20-60°C for 12-24 hours, centrifuge or filter to remove the precipitate, and adjust the pH value of the hydrolyzate to 4-6; Method 11: Use Ca(OH) ₂ to adjust the pH value of the hydrolyzate to 9-11, react in a water bath at 20-60°C for 12-24 hours, adjust the pH value of the hydrolyzate to 4-6, and then add activated carbon to react for 5min- 30min, centrifuge or filter to remove precipitate; Method 12: Use Ca(OH) ₂ to adjust the pH value of the hydrolyzate to 4-6, centrifuge or filter to remove the precipitate, add laccase and react in a water bath at 30-60°C for 12-24 hours; Method 13: Use ammonia water to adjust the pH value of the hydrolyzate to 9-11, react in a water bath at 20-60°C for 12-24 hours, adjust the pH value of the hydrolyzate to 4-6, then add activated carbon to react for 5min-30min, centrifuge or filter to remove the precipitate; Method 14: Use ammonia water to adjust the pH value of the hydrolyzate to 4-6, centrifuge or filter to remove the precipitate, add 10% laccase with an enzyme activity of 2.75 U/mL, and react in a water bath at 30-60°C for 12-24 hours; (3) Take the above-mentioned detoxified hydrolyzate as the carbon source of the medium, add 0.1-2% nitrogen source, and use it as the medium after sterilizing at 121°C for 15-20 minutes; add acetic acid with an inoculation amount of 5%-10% bacillus or glucose oxidizing bacillus, shake culture at 25-30°C, 160-250rpm or static culture at 25-30°C for 6-25 days to obtain bacterial cellulose; wherein, the nitrogen source is yeast extract or peptone. 2. A method for producing bacterial cellulose by using corn stalks according to claim 1, characterized in that: in the step (2), add activated carbon with a mass percentage of 1%-6% and stir at room temperature for 5-10min . 3. A method for producing bacterial cellulose by using corn stalks according to claim 1, characterized in that: the culture medium in the step (3) uses a detoxified hydrolyzate as the carbon source of the culture medium, at a rate of 7-30g/ The amount of reducing sugar in L. The hydrolyzate is prepared into a fermentation medium, which contains 0.1-1% yeast extract, 0.1-0.5% peptone, and the pH value of the medium is 5.0; the amount of reducing sugar is measured by glucose.

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